INTRODUCTION

The American mayapple (Podophyllum peltatum L.; Berberidaceae) is a
rhizomatous perennial species found throughout eastern North America. The species
has received attention recently due to pharmaceutical compounds found in its
leaves. Podophyllotoxin, along with a-peltatin, and
b-peltatin, are aryltetralin lignans known to have
biological activity such as anti-cancer, anti-fungal, anti-viral, anti-mitotic,
and immunostimulatory properties (Rahman et al. 1995; Goel et al. 1998; Imbert
1998; Pugh et al. 2001). Podophyllotoxin is also used as a precursor in the
semi-synthesis of etoposide, teniposide, and etopophos, three drugs used in
chemotherapy (Stähelin and von Wartburg 1991; Giri and Narasu 2000).

Podophyllotoxin is currently available from the Indian mayapple [(Podophyllum
emodi Wall., Berberidaceae (syn. P. hexandrum Royle)], a species
native to specific regions of the Himalayas. Rhizomes and roots of the Indian
mayapple contain up to 53.5 mg×g-1 podophyllotoxin on a dry
weight basis compared to 14.7 mg×g-1 for rhizomes of the American
mayapple. However, leaves of the American mayapple contain up to 55.9 mg×g-1
podophyllotoxin on a dry weight basis (Canel et al. 2001; Moraes et al. 2001).
Because leaves are a renewable resource, it may be possible to establish commercial
plantings of this species. Rhizomes could be left undisturbed while leaves could
be harvested annually. In addition, commercial plantings could reduce harvest
pressure on the Indian species, which was declared endangered due to over-exploitation
of wild populations (Foster 1993; Nautiyal 1996; Giri and Narasu 2000; Rai et
al. 2000).

It has been shown that podophyllotoxin content in leaves of American mayapple
is highly variable, and some wild populations have very low content. Wildcrafting
(that is, harvesting of plant materials from wild populations) would lead to
inconsistent results and possibly poor drug yield (Bastos et al. 1996; Moraes
et al. 2001). Commercial plantings, in contrast, would rely on selection and
propagation of clonal populations with high podophyllotoxin content. Successful
propagation of American mayapple using in vitro techniques has been reported
(Sadowska et al. 1997; Moraes-Cerdeira et al. 1998), but most reports of vegetative
propagation of Podophyllum species have been limited to the Indian mayapple.
Results have been highly variable using rhizome cuttings. From 34% to 98% of
rhizome cuttings 2.0 to 2.5 cm in length and planted from May to July in India
were successful in establishing plants (Badhwar and Sharma 1963; Choudhary et
al. 1998). Nadeem et al. (2000) found 70% of rhizome cuttings treated with 100
mm IBA produced roots compared to only 30% for untreated cuttings. Troup (1915)
estimated that plants grown from rhizome cuttings would require 12 years to
attain marketable size.

Rhizome structure of American mayapple is distinctly different from that of
the Indian mayapple. Reports about vegetative propagation of American mayapple
were not found; therefore, the purpose of research presented in this report
was to identify types of rhizome cuttings suitable for establishing plantings
of American mayapple under field conditions. In addition, the purpose was to
compare fall versus spring planting.

METHODOLOGY

Mayapple rhizomes of known podophyllotoxin content were harvested from a native
population in Oxford, Mississippi on Oct. 12, 2000 and Feb. 20, 2001. At each
harvest, rhizomes were divided into segments to create three types of propagules:
two-node segments comprised of a single terminal node, an adjacent dormant node,
and several cm of rhizome tissue (T+D); one-node segments comprised of a single
terminal node with 3 to 5 cm of rhizome tissue (T); and one-node segments comprised
of a single dormant node with 3 to 5 cm of rhizome tissue extending from each
side of the node (D). Only well-rooted propagules were used, though T segments
generally had shorter and fewer roots than T+D and D segments.

Propagules were transplanted to raised beds located in Verona, Mississippi,
soil type Ora fine sandy loam (fine-loamy, siliceous, thermic, typic Fragiudults),
one day after harvest. Raised beds were prepared in fall 2000 with a press-pan-type
bed shaper and spaced 1.8 m apart, center to center. Beds were formed 15 cm
high and 0.8 m wide across the top, and drip irrigation tubing was installed
in the center of the bed. Each experimental unit consisted of 30 propagules
of a single type arranged in two parallel rows spaced 30 cm apart and 1.5 m
in length. Propagules were placed 10 cm apart and 45 cm deep within each
row and covered with soil. Care was taken to orient propagules with roots down
and nodes up. Beds were then mulched with a 10 cm layer of wheat straw. Irrigation
was seldom needed during the spring of 2001 due to timely and frequent precipitation.
Plantings were side dressed once with ammonium nitrate fertilizer applied at
the rate of 22 kg·ha-1 N in May. Weeds that emerged through
the mulch during growth and senescence of the mayapple crop were removed by
hand.

A 2×3 factorial arrangement of treatments (three levels of propagule
type and two levels of planting time) was used in a randomized complete block
design with four replications. Date of shoot emergence, days to senescence,
and plant height of the mature mayapple plants were recorded during the spring
of 2001. Leaves were removed from the plants to measure leaf area after plants
began to senesce. Leaf area was measured using a LI-3100 Area Meter (LI-COR,
Lincoln, Nebraska). Data were analyzed with Statistical Analysis System for
PC using the General Linear Model procedure (SAS Institute Inc., Cary, North
Carolina). The Mixed procedure was used for the height data because of an unequal
number of subsamples.

RESULTS AND DISCUSSION

Mayapple shoots emerged in March, grew in April and May, and senesced in June
(Fig. 1). Propagule type had a greater effect on shoot emergence than planting
time. Propagules T+D and T exhibited higher percentages of emergence than propagule
D regardless of planting time (Table 1). However, a significant interaction
occurred between propagule type and planting time. Spring-planted T+D exhibited
a higher percentage of emergence than fall-planted T+D while spring-planted
D exhibited a lower percentage compared fall-planted D. Propagule T was not
affected by planting time.

Fig. 1. Emergence and senescence of mayapple (P. peltatum) shoots
in spring of 2001. Relative number of shoots emerged from three types of rhizome
propagules: two-node rhizome segment with a terminal and dormant node (T+D),
one-node rhizome segment with single terminal node (T), or one-node rhizome
segment with single dormant node (D). Values are means ±se of four replications
with up to 30 subsamples per replication. Data from spring- and fall-planted
treatments were pooled.

Propagule type and planting time affected leaf size (cm2 per leaf).
Propagules T+D and D consistently produced leaves about twice the size of propagule
T, and propagules planted in the spring produced leaves about twice the size
of those planted in the fall (Table 1).

Table 1. Shoot emergence, leaf area, leaf size, and plant height of
three types of mayapple (P. peltatum) propagules planted in fall 2000
or spring 2001. Propagules were two-node rhizome segments consisting of a terminal
node and a dormant node (T+D), one-node rhizome segments consisting of either
a single terminal node (T), or a single dormant node (D). About 5 cm of subtending
rhizome material remained attached to each node.

Propagule type

Shoot emergencez
(%)

Leaf areaz
(cm2·m-2)

Leaf sizezx (cm2)

Plant heightzw (cm)

Fall

Spring

Mean

Fall

Spring

Mean

Fall

Spring

Mean

Fall

Spring

Mean

T+D

85

101y

93

679

1484

1082

32

59

45

11

13

13

T

76

88

82

275

842

558

14

37

26

10

13

12

D

54

18

36

467

285

376

34

66

50

12

12

12

Mean

72

69

474

871

27

54

11

13

Significancev

Propagule type

0.000

0.0001

0.0059

0.1485

Planting time

0.5081

0.0001

0.0001

0.0114

Type × time

0.0001

0.0003

0.8167

0.1458

zValues are means of four replications with 30 propagules in each
replication. Not all propagules emerged.yMore than one shoot can arise from each propagule. Thus, values
greater than 100% are possible.xLeaf size calculated from leaf area divided by number of emerged
shoots.wPlant height measured from soil surface to highest point of the
plant.vP values for propagule type and planting time effects.

Propagule type and planting time also affected leaf area (cm2·m-2).
The combined effect of greater emergence and larger leaf size resulted in propagule
T+D exhibiting significantly greater leaf area than propagules T or D. In addition,
spring-planted T+D exhibited significantly greater leaf area than fall-planted
T+D (Table 1). Spring-planted T also exhibited significantly greater leaf area
than fall-planted T, and this was due to the larger leaf size of spring-planted
compared to fall-planted T. Small leaf size of fall-planted T and low percentage
of emergence of spring-planted D resulted in lower leaf area for each of these
treatment combinations and contributed toward the significant interaction effect
for leaf area.

Planting time had a small but significant effect on plant height. Spring-planted
propagules were slightly taller than fall-planted propagules.

Overall, spring-planted T+D propagules produced the greatest leaf area and
appeared to offer greater potential yields of leaf tissue compared to the other
treatment combinations. T+D propagules were larger than the other propagules,
which resulted in greater growth. Propagule mass, however, was not the only
factor influencing leaf area. Spring-planted T produced slightly more leaf area
than fall-planted T+D. Propagules T+D and T appeared to benefit from having
well developed terminal buds that were able to emerge in the spring in greater
numbers than propagule D. Despite the greater leaf size of D, a lower percentage
of emergence resulted in lower leaf area.

Physiological factors characteristic of mayapple rhizomes may have influenced
the low percentage of emergence of propagule D. First, mayapple rhizomes exhibit
strong apical dominance. Regardless of the number of nodes along the length
of a rhizome system, only the terminal node produces a shoot in most cases.
When a node is severed from the rhizome system by insect, animal, or human activity,
the node is released from apical dominance and produces a shoot. It is not known,
however, how long apical dominance persists in rhizome segments after being
severed from the rhizome system. The results of this study show that spring-planted
D exhibited a lower percentage of emergence than fall-planted D and this suggests
that apical dominance was more persistent in the spring-planted propagules than
in the fall-planted material. Second, mayapple rhizomes in the wild are dormant
during the winter months and we have found that exposure to low temperature
is required to break this dormancy (unpubl. data). Propagules used in this study
were exposed to naturally occurring cold temperatures either after they were
transplanted, i.e. fall-planted propagules that overwintered in raised beds,
or before they were harvested, i.e. spring-planted propagules that overwintered
in the wild. We have observed that dormant node segments appear to require more
chilling than segments with a terminal node. Chilling may have influenced fall-planted
propagules, which received chilling after being severed from the parent rhizome,
differently than spring-planted propagules, which received chilling while remaining
attached to the parent rhizome.

Reports have shown that rhizome diameter is correlated with plant size such
that larger rhizomes produce larger plants. Propagules used in this study were
not sorted by rhizome diameter but were assigned randomly to each experimental
unit. Differences in leaf size in this study may be an indication of the effect
of propagule vigor. Spring-planted T+D and D exhibited significantly greater
leaf size than the other treatment combinations. In a similar manner, spring-planted
T+D and T exhibited slightly greater plant height than the other treatment combinations.
These measures of plant growth may be indirect indications of the superior vigor
of these propagules and of the optimum time of harvest and planting. The American
mayapple is a slow-growing plant and we report here first-year observations
from an ongoing study.

REFERENCES

Badhwar, R.L. and B.K. Sharma. 1963. A note on the germination of Podophyllum
seeds. Indian Forester 89:445447.